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ISSN: 2320-5407 Int. J. Adv. Res.

5(2), 1743-1749

Journal Homepage:

Article DOI:10.21474/IJAR01/3336

Asha D1, Dr. Lizzy Mathew1, R. Shankar Iyer2, Sayana P. S.2 and Lekshmi V. Bose3.
1. Department of Botany, St. Teresas College, Ernakulam, Kerala, India.
2. New Udaya Pharmacy & Ayurvedic Laboratories, Cochin, Kerala, India.
3. School of Environmental Sciences, M.G. University, Kottayam, Kerala, India.
Manuscript Info Abstract
Manuscript History Rosemary (Rosmarinus officinalis L.), a spice and medicinal herb of
Lamiaceae family with a characteristic aromatic smell, is widely used
Received: 26 December 2016 around the world and accepted as one of the spices with the highest
Final Accepted: 29 January 2017 antioxidant activity. The aim of this research was to identify and isolate
Published: February 2017
the unknown saponins from the leaves of R. officinalis L. using
HPTLC, HPLC, UV, FTIR and HR-LCMS techniques. The saponins
Key words:- were identified by High Performance Thin Layer Chromatography
Rosmarinus officinalis L., Rosemary, (HPTLC) and confirmed by HPLC. On the basis of spectral data
Saponins, HPTLC, HPLC, UV, FTIR, analysis, the structure of the new saponin isolated by HPTLC from
methanol extract of leaves of R. officinalis L. has been formulated by
UV, FTIR and HR -LCMS spectral analysis as 1alpha-hydroxy-18-(4-
D3/1alpha-hydr. This is a new saponin isolated from R. officinalis L.
and being reported for the first time.
Copy Right, IJAR, 2017,. All rights reserved.
Rosmarinus officinalis L.(Lamiaceae), Rosemary, is a perennial herb native to the Mediterranean region but is
widely distributed in many parts of the world. It grows as a shrub or herbaceous plant with about 0.8 to 2m height
(Atik bekkara et al., 2007). This plant prefers dry and arid regions, hills and low mountains, calcareous, shale, clay
and rocky substrates (El Amrani et al., 1997). The herb R. officinalis L. has been used as a food spice and medicine
since ancient times. The fragrance of the leaf has been said to enhance memory. Rosemary oil was applied to the
skin to treat muscle and joint pain and taken internally to promote abortions. Its use since ancient times in traditional
medicine is justified by its antiseptic (Bult et al., 1985), antirheumatic (Makino et al., 2000), anti-inflammatory,
antispasmodic (Juhas et al., 2009; Beninca et al., 2011), antimicrobial and anti-hepatotoxic properties (Stefanovits-
Banyai et al.,2003). Its appreciation as a spice for seasoning and food preservation (Arnold et al., 1997) is supported
by a very high antioxidant activity (Wang et al., 2008). This antioxidant activity of R. officinalis L. is due to its
phenolic compounds including: carnosic acid, carnosol, rosmarinic acid and hydroxycinnamic acid ester (Inatani et
al., 1983). Aerial parts of R. officinalis L. are orally used to relieve renal colic and dysmenorrhoea (Gonzalez-
Trujano et al., 2007). Recent research shows that R. officinalis L. extracts possess strong anticancer properties
(Vassiliki et al., 2013).

Nowadays market demand of the plant is growing, as it is used in several medicinal products. R. officinalis L. is
indigenous to South Europe and Asia but it is also cultivated in Mediterranean basin and India (WHO, 2007). It is

Corresponding Author:- Asha D. 1743

Address:-Department of Botany, St. Teresas College, Ernakulam, Kerala, India.
ISSN: 2320-5407 Int. J. Adv. Res. 5(2), 1743-1749

used as carminative, rubifacient, stimulant and as flavouring agent for liniments, hair lotions, inhaler, soaps and
cosmetics (Kokate et al., 2010). Rosemary leaves have many traditional uses based on their antibacterial and
spasmolytic actions. Used orally for the treatment of dyspeptic complaints (British Herbal,1996), and in external
applications for supportive management of rheumatic complaints and circulatory disorders (Blumenthal,1998). It is
used as a cholagogue, diaphoretic, digestant, diuretic, emmenagogue, laxative and tonic (Bedevian,1994;
Farnsworth, 2005) also used in the management of headache, menstrual disorders, nervous menstrual complaints,
tiredness, defective memory, sprains and bruises (Hagers, 2003; Asia et al.,2013)

It is described in cases of congestion of the liver, inflammation of the gall bladder, gastric lavage, in some cases of
jaundice, fatigue, physical and intellectual weakness following the diseases debilitating to the body, migraine,
dizziness, palpitations, jittering, strikes, heartburn, carminative and as an antiseptic (Antoine, 1998). Many
compounds have been isolated from R. officinalis L. including flavones, diterpenes, steroids, and triterpenes.

Saponins are natural high-molecular-weight glycosides of triterpene or steroids with a very wide distribution in the
plant kingdom (Hostettmann and Marston, 1995). Saponins exhibits a range of biological activities (Oleszek and
Marston, 2000) which include, anticholesterolemic (Oakenfull, 1981), anti-inflammatory, anti-parasitic and antiviral
(Just et al.,1998; Traore et al.,2000). Saponins are also effective against drug-resistant cancer cells (Cheung et
al.,2005). The great structural diversity of saponins, novel bioactivities which is relevant to the pharmaceutical
industry, the challenges of identification, are now opening new opportunities or newer trends for exploitation of
novel saponins. Hence the aim of the current investigation was to identify and characterize active saponins from the
leaves of medicinally important plant R. officinalis L.

Materials and Methods:-

The fresh plants of R. officinalis L. were collected from Ooty, Tamil Nadu, India. The plant materials were
identified and authenticated by Dr. K.V. George, Emeritus Scientist (KSCSTE), Department of Botany,
St.Berchmans College, Changanacherry, Kerala. The voucher specimen (Voucher No.N/PG/074) is deposited in the
herbarium of New Udaya Pharmacy & Ayurvedic Laboratories, Cochin, Kerala, India.

Sample preparation:-
About 10 gm of the powdered sample was taken in a thimble and extracted with 250 ml methanol in a soxhlet
apparatus. The extract was then concentrated in a Rotary Vaccum Evaporator to a volume of 30 ml and stored in
small air tight brown bottles. This methanolic extract was used for the screening and isolation of compound.

High Performance Thin Layer Chromatography (HPTLC):-

HPTLC is a flexible, reliable, and cost-efficient separation technique ideally suited for the analysis of botanicals and
herbal drugs. HPTLC studies were carried out using the method described by Wagner et al. (1996). Methanol extract
of the selected plant was subjected to HPTLC (CAMAG, Switzerland) analysis. A Camag HPTLC instrument
consisting of Linomat V automatic spotter equipped with a 100 L syringe connected to a nitrogen cylinder,
Scanner-III, twin-trough developing chambers, and viewing cabinet with dual wavelength UV lamps (Camag,
Muttenz, Switzerland) were used. Before analysis, HPTLC plates were cleaned by predevelopment with methanol
and activated at 110C for 5min for solvent removal. Plant extract were spotted on a silica gel 60F 254 (Merck,
Germany) TLC plate. The plate was air dried and then developed by using the solvent system Chloroform: Acetic
acid: Methanol: Water (6.4:3.2:1.2:0.8) (v/v/v/v) as mobile phase in a CAMAG- twin-trough glass chamber
(20x10x4) previously saturated with mobile phase vapour for 20 minutes. After developing the plate, it was dried
and scanned using Scanner 3 (CAMAG, Switzerland) at 275 nm using WinCATS software. Chromatograms were
evaluated before and after spraying with Anisaldehyde sulphuric acid reagent. After derivatization, plate was dried
in hot air oven for 5 minutes at 105 C and viewed under UV at 366 nm.

High Performance Liquid Chromatography (HPLC):-

High Performance Liquid Chromatography was used to analyse the isolated fraction obtained from HPTLC. Sample
was dissolved in HPLC grade methanol in concentration of about 1-10g/ml, 20l of the solution was injected in the
column RP-C18 and analyzed by PDA detector. The wavelength range was 250 - 500nm. Thermo HPLC system
consisted of Quaternary gradient pumps (LC 10ATvp), Photodiode Array (PDA) and detector (SPD M10Avp)
with built-in system controller. The analysis was performed on a 25 cm x 4.6 mm, 5 m particle size CNW, Athena
C18-WP column. The data acquisition was done on Chrom Quest 5 software. The isolated compound from HPTLC
was analyzed by using Methanol: Acetonitrile (95:5) as mobile phase.

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Characterization of isolated compound:-

The isolated compound was characterized by UV, FTIR and HR-LCMS analysis

UV Spectroscopy:-
The absorbance of the isolated compound was read using one cm cell in a UV Vis - NIR spectrophotometer
(Varian, Cary 5000, Netherlands). The instrument have a spectral range of 175 nm to 3300 nm, wavelength
accuracy of 0.1 nm (UV Vis), 0.4 nm (NIR), wavelength reproducibility of 0.025nm and a limiting resolution
of 0.05nm(UV-Vis), 0.2nm(NIR).The maximum range of absorbance of isolated compound in the methanolic
solution was noted by comparing it against HPLC grade methanol as a blank. Separated components (1 mg each)
were dissolved in methanol and recorded the spectrum in the range of 200 to 500 nm using a UV double beam

Fourier Transform Infra Red spectrometer (FTIR):-

FTIR analysis was carried out using Thermo Nicolet, Avatar 370 spectrophotometer system, which was used to
detect the characteristic peaks and their functional groups. The spectral range was between 4000-400 cm-1 and
resolution was 4cm-1with KBr beam splitter, DTGS Detector and HATR Assembly for convenience of
measurement. The finger print region extended between 400 1600 cm -1. The spectrum of the isolated compound
was elucidated against a blank of HPLC grade methanol.

High Resolution Liquid Chromatograph Mass Spectrometer (HRLCMS):-

Further structural analysis was aided with HR-LCMS (High Resolution liquid chromatography-mass spectrometry)
with a mass spectrometer using High Resolution. Liquid chromatography coupled with mass spectrometry (LC/MS)
is a powerful technique for the analysis of complex botanical extracts. HPLC is efficient in separating chemical
compounds in a mixture, and MS provides abundant information for structural elucidation of the compounds. The
LCMS analysis provides the molecular weight information for the components of the extract. MS dissociations give
further structural information on the target compounds (Chen et al., 2007). Therefore, the combination of HPLC and
MS facilitates rapid and accurate identification of chemical compounds in medicinal herbs, especially when a pure
standard is unavailable. The HRLCMS analysis was performed using Agilent Technologies, USA, model 1290
Infinity UHPLC System, 1260 infinity Nano HPLC with Chipcube, 6550 iFunnel Q-TOFs. The mass range is
between 50-3200 amu, resolution is 40000 FWHM, high mass accuracy typically less than 1ppm, sensitivity 1 pg.
reserpine S/N 100:1, direct infusion for mass analysis (MS, MS /MS), binary nano HP- LC system with mass as
detector. The analytical column was an octadecylsilane C18, 250 x 4.6 mm ID, 5 m particle size protected by a
compatible guard column. For the characterization of isolated compound the HPLC method was same as that used in
HPLC with CNW, Athena C18-WP column.

High Performance Thin Layer Chromatography (HPTLC):-
HPTLC of methanol extract of R.officinalis L. was carried out to confirm its nature by analyzing TLC
chromatograms and to isolate active saponin ingredients from the extract. TLC of methanol extract of R. officinalis
L. revealed the presence of 5 compounds (corresponding to 5 spots) having Rf values of 0.26, 0.42, 0.50, 0.59and
0.68 respectively when a solvent phase of Chloroform: Acetic acid: Methanol: Water (6.4:3.2:1.2:0.8) was used
(Fig:1). Compound having Rf of 0.59 was most prominent and showed clear spot (orange spot) when sprayed using
Anisaldehyde sulphuric acid reagent. Hence, this particular spot was selected for further identification and

High Performance Liquid Chromatography (HPLC):-

HPLC of isolated compound from the methanol extract obtained by HPTLC was carried out to confirm its nature by
analyzing HPLC chromatograms. The sharpness of peak, its retention time (Rt min), height and percent area were
recorded. The HPLC chromatogram of isolated compound shown only one peak with prominent significant height
303889 and percent area (> 100%) at the retention time 3.372 (Rt min) (Fig 2).

Characterization of the isolated compound:-

UV & Fourier Transform Infra red Spectophotometry (FTIR):-
UV spectra displayed characteristic absorption band at 260 nm. Data of IR spectrum (KBr, cm-1) exhibited
absorption in the range from 3374.61 cm-1 to 662.59 cm-1. The functional group and the chemical bond

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corresponding to each peak are tabulated in Table 1. The FTIR spectrum indicated the presence of C-H, O-H and
C=O bonds in the isolated compound (Fig 3).

High Resolution Liquid Chromatograph Mass Spectrometer (HR-LCMS) :-

Thus from the FTIR and HR-LCMS chromatogram obtained, the chemical compound isolated from the aerial parts
of R. officinalis L. is identified as a saponin compound with molecular formula:
The molecular structure of the chemical compound in Fig.4.

Fig 1:- HPTLC chromatogram of Rosmarinus officinalis L. After dervatization UV366nm

A.Crude methanolic extract
B. Isolated compound

Fig 2:-HPLC chromatogram of the isolated compound.

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Fig 3:-FTIR spectra of the isolated compound.

Table 1:- Interpretation of IR spectra.

Frequency (cm-1) Functional Group
662.59 -CC-H: C-H bend
1027.17 =C-H stretch
1453.65 C-H bend
1658.25 C=O stretch
2945.83 C-H stretch
3374.61 O-H stretch, H-bonded

Fig 4:- HR- LCMS result of Isolated compound Chemical formula : C28H42O4 Molecular weight : 442.31

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In recent years, although technology and medicine have developed extensively due to decrease in natural richness
and other drawbacks, some countries have made it obligatory to use natural products for many goals (Erturk et
al.,2003). For this reason we have chosen an important medicinal plant R. officinalis L., which is a herb and spice
with incredible medicinal properties. In the above studies, saponins were extracted from the plant by HPTLC and
HPLC and UV, FTIR and HR-LCMS techniques were carried out to investigate unknown saponins present in the
methanol extract.

Renukappa et al. (1999) have applied LC-NMR (liquid chromatography-nuclear magnetic resonance) and LC-mass
and LC-coupled bioassay to determine two anthelmintic dammarane-type triterpenoidal saponins, significantly
active against Caenorhabditis elegans from a crude fraction of Bacopa monniera. Earlier studies on the biological
activities of saponins were limited to crude extracts containing saponins as well as other polar constituents. The
advent of modern sophisticated methods of isolation and structure elucidation has attracted great interest of scientific
community to study structure activity relationships (Garai,2014). Nyberg et al.(2003) also applied solid phase
extraction followed by NMR and MALDITOF mass spectrometry on chromatographic fractions QHA and QH B
of immuno adjuvant active saponins to identify 28 different saponins of Quillaja saponaria. Three new olean type
triterpenoid saponins were isolated by 1D and 2D NMR and MS spectroscopic data from the aerial parts of Eclipta
prostrata ( L.) by Xi et al. (2014). Zhang et al. (2002) identified a new saponin as a ginsenoside-Ro derivative
containing a polyacetylene side chain by spectroscopic means including 1D and 2D NMR.

In conclusion, we can state that the present study revealed the presence of saponins in R. officinalis L. leaves which
were confirmed by various characterization studies. Chemical markers are now applicable in many research areas,
which include authentication of species, identification of adulterants, structure elucidation and purity determination
of medicinal plants. So this isolated saponin can be used as a chemical marker in R. officinalis L. which can be
exploited more in future. Hence an attempt was made to isolate, purify and characterize the unknown saponins
which can be used as markers and can serve as a powerful tool for the standardization of the extracts.

The methanol extract of Rosmarinus officinalis L.(Rosemary) revealed the structure of compound as 1alpha-
hydroxy-18-(4-hydroxy-4-methyl-2-pentynyloxy)-23,24,25,26,27-pentanorvitamin D3/1alpha-hydr. and is found to
be a Saponin moiety which can be used as marker compound. Further studies need to be conducted for its
pharmacological activity.

The authors are grateful for the support of St.Teresas College, Ernakulam, Kerala. The authors wish to thank the
authorities of New Udaya Pharmacy & Ayurvedic Laboratories, Cochin, Kerala for providing facility to carry out
this Research. Authors wish to thank Botanist Dr. K.V. George, Emeritus scientist (KSCSTE), Department of
Botany, St.Berchmans College, Changanacherry, Kerala for helping in the identification and authentication of the

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